In studies concerning taxonomic classification, genetic analysis, or the physiology of catabolism and its regulation, it is common practice to use colorimetric indicator plates to facilitate the isolation or identification of microbial strains. Conventionally, however, the color differences generated with the indicators are based on pH changes. That is, the color change that differentiates the microorganisms is based on the production or consumption of acid by the microorganisms. For this reason the method has primarily found utility in testing for microorganisms that catabolize a sugar or the like to produce acid. Hence, the utility is quite restricted in that it is not applicable to the numerous strains of microorganisms that are characterized by their ability to catabolize amino acids, fatty acids, or other types of compounds with no resultant production of acid or other cause for change in pH. Also, the colorimetric technique based on changes in pH can, in many instances, produce ambiguous results. For example, often the acidic compounds initially produced are then further oxidized thereby resulting in a gradual continued change in the pH and causing the color to fade. Further, since the change in color is predicated solely on change in pH and since the change in pH caused by the microorganisms is often only slight, the technique is satisfactory only where the total chemistry of the system is such that no changes in the pH can occur other than by way of the metabolic behavior of the microorganisms.
Briefly, what I have discovered is that a far more easily controlled, more sensitive, and less restrictive way to test and identify microorganisms is feasible by a composition wherein the change in color is predicated not on a change in pH and the presence of a pH indicator but rather on an oxidation-reduction reaction which involves an oxidation-reduction indicator and which results from the catabolic behavior of the particular microorganism or type of microorganism for which the test is being made. More specifically, the preferred embodiments of the present invention involve a color change by way of the reduction of a tetrazolium compound to a formazan compound. The reducing agent causing the reduction reaction is or results from one or more of the catabolites produced during the catabolism of a test substrate compound included in the composition. Further, and more specifically with respect to the preferred embodiments of the present invention, the compositions used to identify or test the microorganism or strain of microorganism include: (1) a tetrazolium compound which undergoes a change in color upon reduction to a formazan compound; (2) a test substrate compound, i.e. a biodegradable compound which, if catabolized by the microorganism, will produce a catabolite engendering reduction of the tetrazolium compound; (3) a buffer to maintain the pH of the composition at a level which enables microorganism culture growth but which does not itself cause or prevent reduction of the tetrazolium compound to the colored formazan compound; and (4) nutrient for the microorganism or strain of microorganism in a concentration sufficient to induce culture growth without itself causing or resulting in reduction of the tetrazolium compound. Any one such composition is, by way of the particular test substrate compound it includes, useful for the colorimetric testing of a given microorganism or strain of microorganism. However, by using a series of such compositions, each with a different test substrate compound, a culture can be tested so as to identify the microorganism strain in the culture, this by way of the presence or absence of color changes in the various compositions in which the culture is allowed to grow. Hence, the invention further comprehends a test plate, or container, having separate recesses or compartments each of which contains a different such composition such that when a culture of unknown microorganism content is grown while in contact with each of the various compositions, there is indication of the particular microorganisms or microorganism strain in the culture by way of change in color or absence of change in color in the various compositions. The compositions can additionally contain some agar or the like to provide a gel or gel-like consistency for convenient growth of the microorganism cultures on the surface of the compositions.